The overall goal of this Program Project is to develop novel, safe, and effective M. tuberculosis (Mtb)- derived vaccines for the prevention of tuberculosis (TB). Project 3 will focus on investigating the roles of two components ofthe tuberculous granuloma - B cells and tumor necrosis factor (TNF) - in their ability to augment Immunogenicity of anti-TB vaccines. The proposed program derives from data generated in the last funding period. The B cell-deficient )j.MT mouse studies revealed that these lymphocytes are required for the optimal control of acute TB. In chronic infection, T cell response (proliferation and IFNy production) in ^iMT mice Is remarkably altered. Studies using mice deficient in specific Fey receptors (FcyR) have provided evidence that immune complex (IC) can modulate anti-TB cellular immunity. These are compelling evidence that B cells can regulate T cell response in TB infection. Further, a potent M. smegmatis-derwed vaccine IKEPLUS, while highly effective in immunizing wildtype mice against Mtb challenge, failed to protect B cell- deficient animals. Project 3 will characterize the roles of B cells in the development of anti-TB immunity and to test the hypotheses that: i) Specific functions of B cells contribute significantly to the host immune response to Mtb;ii) B cells are required for the development of effective T cell response in TB, including the CD4 T cell memory compartment;iii) B cells are essential for the development of optimal vaccine- engendered protection;and iv) IC can be exploited to augment anti-TB immunity. In the TNF study, we have identified Mtb cosimd clones capable of downregulating macrophage TNF production. We will test the hypothesis that disruption of the TNF-downregulating genes will generate strains that are able to augment macrophage TNF production during infection, thereby enhancing immunogenicity by virtue of their ability to promote phagosome maturation and apoptosis, two processes that can augment T cell immunity. In sum. Project 3 seeks to identify components of the B cell response and TNF-downregulating Mtb genes that can augment immunogenicity of vaccine candidates. These components, when identified, will be incorporated into existing safe and effective Mtb-derived platforms (developed In the last funding period) to further enhance the efficacy of candidate anti-TB vaccines, the overall goal ofthe P01. Thus, there will be close interactions between Project 3 and the other components ofthe Program Project. Finally, the Animal, Flow Cytometry, and Administrative Cores will all be utilized extensively by the program proposed in Project 3. RELEVANCE (See instructions): Project 3, an integral part of the POl, seeks to identify components of B cell immunity and TNF- downregulating genes that can be targeted to enhance host Immunity against Mtb. Identificaion of such factors will guide the rationale design of safe and effective Mtb-derived attenuated vaccine candidates with high level of immunogenicity that can effectively engender protection against the tubercle bacillus.